Diacetyl by Wine Making Lactic Acid Bacteria
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Agric. Biol. Chem., 49 (7), 2147-2157, 1985 2147 Transformation of Citric Acid to Acetic Acid, Acetoin and Diacetyl by Wine Making Lactic Acid Bacteria Yoshimi Shimazu, Mikio Uehara and Masazumi Watanabe Food Research Laboratory, KikkomanCorporation, 399 Noda, Noda-shi, Chiba 278, Japan Received January 18, 1985 Adecrease in citric acid and increases in acetic acid, acetoin and diacetyl were found in the test red wine after inoculation of intact cells of Leuconostoc mesenteroides subsp. lactosum ATCC27307, a malo-lactic bacterium, grownon the malate plus citrate-medium. Citric acid in the buffer solution was transformed to acetic acid, acetoin and diacetyl in the pH range of 2 to 6 after inoculation with intact cells of this bacterial species. It was concluded that citric acid in wine making involving malo- lactic fermentation, at first, was converted by citrate lyase to acetic and oxaloacetic acids, and the latter was successively transformed by decarboxylation to pyruvic acid which was subsequently converted to acetoin, diacetyl and acetic acid. Both the activities of citrate lyase and acetoin formation from pyruvic acid in the dialyzed cell- free extract were optimal at pH 6.0. Divalent cations such as Mn2+ , Mg2+ , Co2 + and Zn2 + activated the citrate lyase. The citrate lyase was completely inhibited by EDTA,Hg2+ and Ag2 +. The acetoin formation from pyruvic acid was significantly stimulated by thiamine pyrophosphate and CoCl2, and inhibited by oxaloacetic acid. Specific activities of the citrate lyase and acetoin formation were considerably variable amongthe six strains of malo-lactic bacteria examined. Someactivities of irreversible reduction of diacetyl to acetoin were foundin the cell-free extracts of four of the malo- lactic bacteria strains and the optimal pH was 6.0 for this activity of Leu. mesenteroides. Malo-lactic fermentation (MLF) in wine lished on the correlation between the levels of making is the process of conversion of malic acetoin and diacetyl in malo-lactic fermented acid to lactic acid and carbon dioxide by the wines and their sensory evaluation.16 ~20) action of certain lactic acid bacteria.1~4) This paper describes the cleavage of citric Furthermore, MLFcontributes not only to the acid to oxaloacetic and acetic acids, and the reduction of high acidity of wines, but also to subsequent formation of acetoin, diacetyl and the enhancement and complexity of wine acetic acid from pyruvic acid by intact cells flavors.5 ~8) or cell-free extracts of malo-lactic bacteria iso- In previous papers9'10) we reported that lated from fermenting wine musts. citric acid in addition to malic acid in must was considerably decomposedwith the occurrence of MLFand the amounts of acetoin in malo- MATERIALS AND METHODS lactic fermented wines increased markedly. Microorganisms. Six bacterial strains isolated from The mechanism of the formation of acetoin malo-lactic fermented musts were used. Leuconostoc and diacetyl by Streptococcus and Leuconostoc mesenteroides subsp. lactosum ATCC27307 (Nonomura 234),21) Leu. infrequens ATCC 27308 (Nonomura 83)21) species in cheese, butter and milk products has and Leu. dextranicum subsp. vinarium 721) were kindly been studied in detail by several workers.ll ~15) donated by Prof. H. Nonomura (Department of On the other hand, no report has yet been Fermentation Technology, Yamanashi University). Leu published, on the mechanismof the transfor- oenos PSU-122) was obtained from Tri Bio Laboratories, mation of citric acid to acetoin and diacetyl in Inc. (U.S.A.). Lactobacillus hilgardii BC2was a generous MLF, although many reports have been pub- gift from the Institut D'oenologie, Universite de 2148 Y. Shimazu, M. Uehara and M. Watanabe Bordeaux, France. Lac. brevis B-9 was isolated from malo- 30°C for 30min, the reaction was stopped by heating in a lactic fermented must at our laboratory and classified by boiling water bath for 5min. The amount of acetoin was us. Leu. mesenteroides subsp. lactosum ATCC27307 was determined by gas chromatography. Oneunit of activity of used throughout to elucidate the transformation me- acetoin formation was denned as the amount of the cell- chanismfor citric acid. free extract required to liberate one /imol of acetoin from Saccharomyces cerevisiae IAM4274, from the Institute the substrate per minute at 30°C under the above of Applied Microbiology, Tokyo University, was used as a conditions. vinification starter. Determination of diacetyl reduction and acetoin dehy- Culture media and cultivation. The modified drogenation. Diacetyl-acetoin reduction was assayed by Nonomura's basal mediumfor lactic acid bacteria con- determining acetoin formed in a reaction mixture consist- tained 20g peptone, 5g yeast extract, 0.2g liver extract, ing of 1 mM diacetyl and 0.14mM NADHin 3.2ml of0.2m 0.2g Tween 80, 0.08g MnCl2, 0.2g MgSO4, 0.5g phosphate buffer (pH 6.0) and 0.3ml of the cell-free KH2PO4, 10g glucose and 5g fructose per liter of de- extract in a total volume of 3.5 ml. Acetoin-diacetyl dehy- ionized water. Ten grams of sodium citrate or sodium l- drogenation was assayed by determining diacetyl formed malate was added to the basal mediumfor pre-adaptation in a reaction mixture (final volume, 3.5ml) containing of the bacteria tested to each organic acid. Citrate and l- 1mMacetoin and 1mMNAD.Both mixtures were in- malate media were respectively adjusted to pH 5.0 and 6.5 cubated at 30°C for 30min. One unit of activity ofdiacetyl with lN-NaOH and autoclaved at 120°C for 20min. reduction or acetoin dehydrogenation was, respectively, Cultivation was carried out at 30°C for 3 days. defined as the amount of the cell-free extract required to liberate one /rniol ofacetoin or diacetyl from the respective Preparation of intact cells and cell-free extracts of lactic substrate per minuteunder the aboveconditions. acid bacteria. The cells of lactic acid bacteria were harvest- ed from the culture broth by centrifugation at 12,000 x g Vinification. Grape must was prepared from Muscat for 30min at 4°C and then washed twice with 0.02m Bailey A grapes (36kg) harvested in 1982 in Yamanashi, potassium phosphate buffer (pH 6.0). The washed cells Japan. The must was treated with 50mgof sulfur dioxide were suspended in a small amount of 0.02m phosphate per liter of must in the form of potassium metabisulfite buffer (pH 6.0). The cells were disrupted at 0°C for 15 min and ameliorated with sucrose to a sugar concentration of with a Branson Sonifier Cell Disruptor 200P. The debris 22%. The prepared must (22 liters) was divided into two was removed by centrifugation at 34,000xg at 4°C for portions (each ll liters) and then each portion was 20min. The supernatant was dialyzed overnight against fermented on the skins at 23~25°C for 10 days. The first the same buffer (pH 6.0) at 4°C and used as the cell-free portion of the must (must a) was subjected to conven- extract. tional vinification without artifical inoculation with cul- For examination of the effects of metal ions on the tures of malo-lactic bacteria during fermentation. Wash- enzyme activity, 5.32g of solid ammoniumsulfate was ed cells (1 x 106 cells/ml) of Leu. mesenteroides cultivated added to 10ml ofa cell-free extract with stirring at pH 6.0. on the malic plus citric acids-medium were inoculated The precipitate collected by centrifugation was dissolved into the second portion of must (must b) on the fifth in 0.02m potassium phosphate buffer (pH 6.0) and dia- day (alcohol content, 10.4 vol%) of fermentation. lyzed overnight against the same buffer at 4°C. Wines A and B respectively obtained from musts a and b were stored at approximately 18°C for 30 days, Assay of citrate lyase activity. Citrate lyase activity was racked, sterile-filtered and finally bottled. assayed by determining acetic acid formed in a reaction mixture containing 5min sodium citrate, 1 mMMgCl2, Analyses. Chemical components of the test wines were 0.2m phosphate buffer (pH 6.0) and 0.3 ml of the cell-free analyzed by the methods of Hennig and Jakob.23) 2,4- extract in a total volume of 3.5ml. The reaction was Dinitrophenylhydrazine (DNPH) derivatives of pyruvic carried out at 30°C for 30min and stopped by the addition and oxaloacetic acids, and acetoin and diacetyl were of0.2ml of6n HC1solution. One unit of citrate lyase was identified by the descending paper chromatographic defined as the amount of the enzymerequired to liberate method used in a previous study.24) one /zmol of acetic acid from the substrate per minute at Citric, acetic, lactic, malic, formic, tartaric, succinic and 30°Cunder the above conditions. galacturonic acids were determined with a carboxylic acid analyzer model S-14 (Tokyo Rikakikai Co., Ltd., Tokyo) Assay of activity of acetoin formation from pyruvic acid. according to the EDC [l -ethyl-3-(3-dimethylaminopropyl) Acetoin formation from pyruvic acid by a cell-free extract carbodiimide hydrochloride] method of Shimazu et al.25) was assayed as follows. To 3.2ml of 0.2m phosphate l- and D-Lactic, and pyruvic and oxaloacetic acids were buffer (pH 6.0) containing 10mMsodium pyruvate, 1 mM assayed enzymatically as described previously.24) Acetoin CoCl2 and 0.5mM thiamine pyrophosphate (TPP) was and diacetyl were determined using a JEOLgas chroma- added 0.3ml of the cell-free extract. After incubation at tograph model JGC-1100 (Japan Electron Optics Transformation of Citric Acid by Wine Making Lactic Acid Bacteria 2149 Laboratory Co., Ltd.).26) Protein in the cell-free extracts Table I. Compositions of Muscat Bailey A Wine was determined by the method of Lowry et al.21) with Inoculated with or without Leu. mesenteroides bovine serum albumin as a standard.